Figure 5.

The influence of EMT on mitotic roundness in MCF‐7 tumor spheroids. a) Confocal images of cross‐sections of MCF‐7 spheroids in PEG–heparin gel after 1 week of growth. Cells were fixed and stained for DNA (DAPI, blue) and F‐actin (phalloidin, red). Mitotic cells were arrested in mitosis through co‐incubation with STC (2 × 10⁻⁶ m) prior to fixation and exhibit condensed chromosomes (see, e.g., cells in green frame). Scale bar: 10 µm. b) Left: zoom of cross‐sections of mitotic cells in (a). Right: For image analysis, the cell outline of the largest cell cross‐section was first determined (white). Roundness of the cell was then calculated using Fiji including the fit of an ellipse (yellow), see Experimental Section. c) Roundness of mitotic cells in spheroids before and after EMT in compliant and stiff hydrogels (Young's modulus of ≈3 or ≈9 kPa, respectively). d) Roundness of mitotic cells in compliant gels with or without inhibition of cortical contractility through the myosin inhibitor Blebbistatin (10 × 10⁻⁶ m). e) Roundness of mitotic cells in compliant gels with or without Rac1 inhibition upon 48 h treatment with 50 × 10⁻⁶ m NSC23766 (NSC) and RhoA activation upon 2 or 48 h treatment with 1 µg mL⁻¹ RhoA activator‐II (RhoA‐II). f) Influence of EMT on spheroid size in compliant and stiff hydrogels. Spheroid size was quantified by the largest cross‐sectional area of the spheroid. g) Influence of myosin inhibition on spheroid size in compliant gels (Blebbistatin treatment for 2 days at 10 × 10⁻⁶ m, see Experimental Section) in pre‐ and post‐EMT conditions. h) Influence of Rac1 inhibition on spheroid size upon 48 h treatment with 50 × 10⁻⁶ m NSC23766 (NSC) and RhoA activation upon 2 or 48 h treatment with 1 µg mL⁻¹ RhoA activator‐II (RhoA‐II) on spheroid size in pre‐ and post‐EMT conditions. (Post‐EMT MCF‐7 cells are referred to as modMCF‐7. Blue‐shaded boxes indicate post‐EMT conditions. Number of mitotic cells analyzed: (c): Compliant gel, MCF‐7 n = 133, modMCF‐7 n = 92, Stiff gel, MCF‐7 n = 93 and modMCF‐7 n = 71. (d): MCF‐7 n = 26, MCF‐7 + Bleb., n = 54, modMCF‐7 n = 58, and modMCF‐7 + Bleb. n = 47. (e): MCF‐7 n = 155, NSC (48 h) n = 192, RhoA‐II (2 h) n = 202, RhoA‐II (48 h) n = 167, modMCF‐7 n = 71, NSC (48 h) n = 35, RhoA‐II (2 h) n = 50 and RhoA‐II (48 h) n = 83. Number of spheroids analyzed: (f): MCF‐7 n = 37, modMCF‐7 n = 34, (Stiff gel) MCF‐7 n = 17, and modMCF‐7 n = 15. (g): MCF‐7 n = 26, MCF‐7 + Bleb. n = 25, modMCF‐7 n = 31, modMCF‐7 + Bleb. n = 33. (h): MCF‐7 n = 40, NSC (48 h) n = 40, RhoA‐II (2 h) n = 40, RhoA‐II (48h) n = 31, modMCF‐7 n = 41, NSC (48 h) n = 39, RhoA‐II (2 h) n = 40, and RhoA‐II (48 h) n = 42. Measurements are representative for at least two independent experiments. ^ns p > 0.05, *p < 0.05, **p < 0.01, ***p < 0.001).